TCF-ALP: a fluorescent probe for the selective detection of Staphylococcus bacteria and application in "smart" wound dressings

Abstract

Alkaline phosphatase (ALP) is an important enzyme-based biomarker present in several bacterial species; however, it is currently undervalued as a strategy to detect pathogenic bacteria. Here, we explore our ALP-responsive colorimetric and fluorescent probe (TCF-ALP) for such applications. TCF-ALP displayed a colorimetric and fluorescence response towards Staphylococcus aureus (S. aureus), with a limit of detection of 3.7 × 106 CFU mL-1 after 24 h incubation. To our surprise, TCF-ALP proved selective towards Staphylococcus bacteria when compared with Enterococcus faecalis (E. faecalis), and Gram-negative P. aeruginosa and E. coli. Selectivity was also seen in clinically relevant S. aureus biofilms. Owing to the high prevalence and surface location of S. aureus in chronic wounds, TCF-ALP was subsequently encapsulated in polyvinyl alcohol (PVA)-based hydrogels as a proof-of-concept "smart" wound dressing. TCF-ALP hydrogels were capable of detecting S. aureus in planktonic and biofilm assays, and displayed a clear colour change from yellow to purple after 24 h incubation using ex vivo porcine skin models. Overall, TCF-ALP is a simple tool that requires no prior knowledge, training, or specialist equipment, and has the potential to overcome issues related to invasive swabbing and tissue biopsy methods. Thus, TCF-ALP could be used as a tool to monitor the early development of infection in a wound and allow for the rapid provision of appropriate treatment for Staphylococcal bacterial infections.

Scheme 1. The use of TCF-ALP for the selective detection of Staphylococcus spp. via alkaline phosphatase dephosphorylation. Images show an ex vivo experiment utilising a PVA-hydrogel encapsulated with TCF-ALP before and after S. aureus NCTC 10788 inoculation.

Fig. 1. UV-Vis spectra of TCF-ALP (10 μM) after 24 h incubation at 32 °C with S. aureus NCTC 10788 (10⁸ CFU mL⁻¹) in 50 mM Tris-HCl buffer, pH = 9.2.

Fig. 2. (A) Fluorescence spectra of TCF-ALP (10 μM) recorded over the course of 24 h upon addition of S. aureus NCTC 10788 (10⁸ CFU mL⁻¹) in 50 mM Tris-HCl buffer pH = 9.2 at 32 °C. (B) Corresponding change in fluorescence (I/I₀) of TCF-ALP (10 μM) upon addition of S. aureus NCTC 10788 (10⁸ CFU mL⁻¹) in 50 mM Tris-HCl buffer pH = 9.2 at 32 °C. Error bars indicate standard deviation (n = 3). (C) Fluorescence spectra of TCF-ALP (10 μM) after 24 h incubation with various concentrations of S. aureus NCTC 10788 (0–10⁹ CFU mL⁻¹) in 50 mM Tris-HCl buffer pH = 9.2 at 32 °C with the dotted line representing 10¹⁰ CFU mL⁻¹, and (D) corresponding sensitivity graph. (X-intercept 6.568 = 3.70 × 10⁶ CFU mL⁻¹; Y = 14.45X–94.88; R² 0.9759). All experiments used λ_ex = 542 (bandwidth 15) nm and λ_em = 606 nm.

Fig. 3. Selectivity bar chart of TCF-ALP (10 μM) in 50 mM Tris-HCl buffer pH = 9.2 after 1 h incubation with various bacterial strains (10⁸ CFU mL⁻¹) at 32 °C. λ_ex = 542 (bandwidth 15) nm. λ_em = 606 nm. Error bars indicate standard deviation (n = 3).

Fig. 4. (A) Fluorescence spectra of TCF-ALP (10 μM) after 24 h incubation with biofilms of S. aureus NCTC 10788 (10¹¹ CFU per membrane) in 50 mM Tris-HCl buffer pH = 9.2 at 32 °C. (B) Corresponding selectivity bar chart. λ_ex = 542 (bandwidth 15) nm. λ_em = 606 nm. Error bars indicate standard deviation (n = 3). (C) Images taken of negative controls (Membrane and Artificial Wound Fluid (AWF) only) and biofilms of S. aureus NCTC 10788 (10¹¹ CFU per membrane) after 0, 1, and 24 h incubation with 10 μM TCF-ALP in 50 mM Tris-HCl buffer pH = 9.2 at 32 °C.

Fig. 5. (A) Images depicting the colour of TCF-ALP-based PVA hydrogels in the presence of a negative control (Membrane and Artificial Wound Fluid (AWF) only) and a S. aureus NCTC 10788 biofilm (10¹¹ CFU per membrane). Images taken hourly at 0–8 h and 24 h and were repeated in triplicate. (B) TCF-ALP-based PVA hydrogels on non-inoculated porcine skin wound model after 24 h incubation. (C) TCF-ALP-based PVA hydrogels on a porcine skin wound model inoculated with S. aureus NCTC 10788 (10 μL of 10⁸ CFU mL⁻¹), and (D) corresponding fluorescence intensities of the hydrogels shown in (B) and (C). λ_ex = 542 (bandwidth 15) nm. λ_em = 606 nm.